Halmer L, Vestner B, Gruss C
University of Konstanz, Department of Biology, Universitätsstr. 10, 78457 Konstanz, Federal Republic of Germany.
J Biol Chem. 1998 Dec 25;273(52):34792-8. doi: 10.1074/jbc.273.52.34792.
Topoisomerases provide the unlinking activity necessary for replication fork movement during DNA replication. It is uncertain, however, whether topoisomerases are also required for the initiation of replication. To investigate this point, we have performed pulse-chase experiments with SV40 minichromosomes as template to distinguish between the initiation and the elongation of replication. Using an unfractionated cytosolic extract as a source of replication functions, we found that the addition of topoisomerases at the initiation step significantly increased the number of active chromatin templates, whereas addition of topoisomerases at the elongation step had only minor effects. Minichromosomes with an extended chromatin structure as well as protein-free DNA required less topoisomerase for effective replication initiation. We could exclude the possibility that topoisomerases enhance the origin binding of T antigen, the SV40 replication initiator, and propose instead that the arrangement of nucleosomes influences the diffusion of supercoils during initial DNA unwinding. Efficient initiation therefore requires a high local concentration of topoisomerases to relax the torsional stress.
拓扑异构酶为DNA复制过程中复制叉移动提供所需的解链活性。然而，复制起始是否也需要拓扑异构酶尚不确定。为了研究这一点，我们以SV40微型染色体为模板进行了脉冲追踪实验，以区分复制起始和延伸。使用未分级的胞质提取物作为复制功能的来源，我们发现，在起始步骤添加拓扑异构酶显著增加了活性染色质模板的数量，而在延伸步骤添加拓扑异构酶只有轻微影响。具有扩展染色质结构的微型染色体以及无蛋白质的DNA在有效复制起始时所需的拓扑异构酶较少。我们可以排除拓扑异构酶增强SV40复制起始因子T抗原与起始位点结合的可能性，转而提出核小体的排列在初始DNA解旋过程中影响超螺旋的扩散。因此，高效起始需要高局部浓度的拓扑异构酶来缓解扭转应力。
